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[Nachruf] Franz Josef Comes
(2015)
The c-MYC proto-oncogene is a regulator of fundamental cellular processes such as cell cycle progression and apoptosis. The development of novel c-MYC inhibitors that can act by targeting the c-MYC DNA G-quadruplex at the level of transcription would provide potential insight into structure-based design of small molecules and lead to a promising arena for cancer therapy. Herein we report our finding that two simple bis-triazolylcarbazole derivatives can inhibit c-MYC transcription, possibly by stabilizing the c-MYC G-quadruplex. These compounds are prepared using a facile and modular approach based on Cu(I) catalysed azide and alkyne cycloaddition. A carbazole ligand with carboxamide side chains is found to be microenvironment-sensitive and highly selective for "turn-on" detection of c-MYC quadruplex over duplex DNA. This fluorescent probe is applicable to visualize the cellular nucleus in living cells. Interestingly, the ligand binds to c-MYC in an asymmetric fashion and selects the minor-populated conformer via conformational selection.
Cardiac arrhythmias are often associated with mutations in ion channels or other proteins. To enable drug development for distinct arrhythmias, model systems are required that allow implementing patient-specific mutations. We assessed a muscular pump in Caenorhabditis elegans. The pharynx utilizes homologues of most of the ion channels, pumps and transporters defining human cardiac physiology. To yield precise rhythmicity, we optically paced the pharynx using channelrhodopsin-2. We assessed pharynx pumping by extracellular recordings (electropharyngeograms--EPGs), and by a novel video-microscopy based method we developed, which allows analyzing multiple animals simultaneously. Mutations in the L-type VGCC (voltage-gated Ca(2+)-channel) EGL-19 caused prolonged pump duration, as found for analogous mutations in the Cav1.2 channel, associated with long QT syndrome. egl-19 mutations affected ability to pump at high frequency and induced arrhythmicity. The pharyngeal neurons did not influence these effects. We tested whether drugs could ameliorate arrhythmia in the optogenetically paced pharynx. The dihydropyridine analog Nemadipine A prolonged pump duration in wild type, and reduced or prolonged pump duration of distinct egl-19 alleles, thus indicating allele-specific effects. In sum, our model may allow screening of drug candidates affecting specific VGCCs mutations, and permit to better understand the effects of distinct mutations on a macroscopic level.
Transport processes across the membrane are essential to ensure survival of every living cell. Therefore, the exchange of membrane impermeable molecules is mediated by specific transport proteins, which are embedded in the lipid bilayer.
One important class comprises secondary active transporters, which couple very efficiently the uphill transport of the main substrate against its concentration gradient to the downhill transport of an additional substrate. These transporters are widely distributed among all kingdoms of life and accomplish many crucial functions. One function is to counteract the deleterious effect of hyperosmotic stress in bacteria. Several members of the BCCT (betaine-choline-carnitinetransport) family of secondary transporters mediate osmostress protection by the accumulation of the compatible solute betaine or its precursor choline (Lamark et al., 1991; Peter et al., 1996; Ziegler et al., 2010). Besides osmo-dependent sodium or proton-coupled symporters, the BCCT family includes few rare representatives of osmo-independent transporters such as the substrate:product antiporter CaiT from E. coli (Jung et al., 2002; Ziegler et al., 2010).
The best-characterized member of the BCCT family is the sodium-coupled betaine transporter BetP from Corynebacterium glutamicum. BetP together with the ABCtransporter OpuA and the H+-solute symporter ProP, became a paradigm for osmoregulated osmolyte transport. Although, all three transporters were extensively studied, the general mechanism of osmoregulation is still far from being understood. Thus, one task of this thesis was to elucidate further the regulatory properties of BetP.
BetP is tightly regulated by osmotic stress and is able to increase its basal betaine uptake activity dramatically upon elevated osmolalities within one second (Peter et al., 1998a). The osmotic stress is sensed by BetP via two stimuli, one is the increase of the internal K+ concentration above a threshold of 220 mM (Rübenhagen et al., 2001), the second is related to a change in the physical state of the membrane (Maximov et al., 2014). So far, several solved crystal structures in combination with functional and computational analysis provided insights into the coupling mechanism of betaine and its co-substrate sodium (Khafizov et al., 2012; Perez et al., 2012). Despite the wealth of data, the precise regulatory mechanism of trimeric BetP is still unclear.
Small molecule drug discovery is strongly supported by biophysical data. In the reach of this thesis, cell free protein expression was used to produce human target proteins for ligand binding assays using Surface Plasmon Resonance spectroscopy (SPR). In the second step the binding and interaction characteristics of small molecules and fragments were analyzed using Nuclear Magnetic Resonance spectroscopy (NMR).
The first target protein was the human acid sensing channel 1 (ASIC1a). ASIC1a was expressed in a cell free expression system based on E.coli lysate. To optimize the expression, several parameters including fusion tags, ion concentrations and different hydrophobic environments were tested.
The adaption of the folding environment for ASIC1a needed more optimization, because it is a very challenging target to express in an in vitro system. Three different expression modes were employed to find a suitable folding environment.
SPR binding studies with ASIC1a were performed with chicken ASIC1a expressed in insect cells. The immobilization of cASIC1a and the used buffer conditions were tested using Psalmotoxin 1, a naturally occurring peptide venom which binds strong to the trimeric form of ASIC1a. Compound characterization experiments were performed with a variety of different ligands including amiloride, a general blocker of the whole ENaC protein family. None of the used ligands showed titration curves that would match a simple 1:1 binding model. The experiments either show no binding signal or signal that could be interpreted as unspecific binding. Even amiloride that should be binding the protein shows no signals that fit a simple binding model.
Another target protein that was investigated is the soluble prolyl cis/trans isomerase Cyclophilin D (or peptidyl prolyl isomerase F – PPIF). This protein is involved in the regulation of the mitochondrial permeability transition pore and therefore a potential drug target to treat neurodegenerative diseases. Small molecule binding was tested with CypD using SPR. Following the kinetic analysis of small molecule ligands, the binding position of different binding fragments was analyzed. These fragments originated from a SPR based fragment screen and gave no co-crystal structures with CypD. Therefore NMR was used to investigate the binding position of these fragments. An analysis of the chemical shift perturbations upon ligand addition revealed that the NMR analysis was in line with the results gathered by x-ray crystallography. The fragments with unknown binding position however, all bind to a specific patch slightly outside the binding pocket.
The ligand CL1 showed a special behavior in the NMR experiments. Upon addition to CypD, it produced large shifts on many signals of the protein, accompanied by a severe line broadening. The shift perturbations were so numerous and large that the spectrum had to be reassigned in complex with the ligand. Triple selective labeling was applied to allow a fast and nearly complete signal assignment. The possibility to use highly sophisticated labeling schemes, is one of the advantages of cell free protein expression. After the assignment of the complex spectrum, the chemical shift perturbations were analyzed and quantified. The residues showing the strongest CSPs are also identified in the crystal structure to be involved in the binding of CL1, giving a consistent picture. The numerous and large shift perturbations, produced by CL1 led to the assumption, that the ligand induces a conformational change in CypD, which is not represented in the co-crystal structure. This conformational change was characterized by a NMR based structure determination. CypD apo yielded a defined bundle, whose folded regions overlap well with the corresponding crystal structure.
For the calculation of the CypD-CL1 complex structure, the sidechain resonances were assigned using an automated assignment approach with the software FLYA. The calculation of the CypD-CL1 complex structure did not result in a defined bundle. While parts of the protein converge in a well folded state, the region around the active site shows no defined folding. Careful analysis of the structure calculation suggests that the problems during structure calculation did not originate from an incorrect resonance assignment, but rather from a lack of NOE crosspeaks. This might be due to a broadening of the corresponding NOE crosspeaks or the coexistence of many different conformations. This leads to the conclusion, that the protein conformation is not defined by the NMR data and could be in a dynamic interchange between multiple structures.
This hypothesis is supported by other observations. The line broadening of the signals in the complex is pronounced in the area around the active site and the substrate binding pocket, hinting to a connection between catalytic activity and protein dynamics. In addition many NMR signals are sensitive to changes in the measurement field strength and the temperature. This field dependent signal splitting suggests dynamic conformational changes in the protein between at least two different conformations on a millisecond timescale.
The current working model is that CL1 binds to CypD and induces the catalytic cycle and the connected conformational changes in CypD. As a result the proline like moiety in CL1 is constantly switching between the cis and the trans conformation. Due to the high affinity of CL1, the inhibitor does not leave the binding pocket after successful catalysis, but stays bound in the pocket stimulating further catalytic cycles. These findings as well as the working model are well in line with data published for Cyclophilin A, another member of the cyclophilin family, thereby supporting the model.
HDAC inhibitors (HDACI), a new class of anticancer agents, induce apoptosis in many cancer entities. JNJ-26481585 is a second generation class І HDACI that displays improved efficacy in preclinical studies compared to the established HDACI SAHA (Vorinostat). Therefore, this study aims at evaluating the effects of JNJ-26481585 on human rhabdomyosarcoma (RMS) and at identifying novel synergistic interactions of JNJ-26481585 or the more common HDACI SAHA with different anticancer drugs in RMS cells. Indeed, we show that JNJ-26481585 and SAHA significantly increase chemotherapeutic drug-induced apoptosis in embryonal and alveolar RMS cell lines, when used in combination with chemotherapeutic agents (i.e. doxorubicin, etoposide, vincristine, and cyclophosphamide) which are currently used in the clinic for the treatment of RMS.
We demonstrate that JNJ-26481585 as single agent and in combination with doxorubicin induces apoptosis, which is characterized by activation of the caspase cascade, PARP cleavage, and DNA fragmentation. Induction of caspase-dependent apoptotic cell death is confirmed by the use of the broad-range caspase inhibitor zVAD.fmk, which significantly decreases both JNJ-26481585-triggered and combination treatment-mediated DNA fragmentation, and in addition completely abrogates loss of cell viability. Importantly, JNJ-26481585 significantly inhibits tumor growth in vivo in two preclinical RMS models, i.e. the chicken chorioallantoic membrane (CAM) model and a xenograft mouse model, supporting the notion that JNJ-26481585 hampers tumor maintenance. Also, in combination with doxorubicin JNJ-26481585 significantly reduces tumor growth in in vivo experiments using the CAM model.
Mechanistically, we identify that JNJ-26481585-induced apoptosis is mediated via the intrinsic apoptotic pathway, since we observe increased loss of mitochondrial membrane potential and activation of the proapoptotic Bcl-2 family members Bax and Bak. Interestingly, we find that JNJ-26481585 triggers induction of Bim, Bmf, Puma, and Noxa on mRNA level as well as on protein level, pointing to an altered transcription of BH3-only proteins as important event for the Bax/Bak-mediated loss of mitochondrial membrane potential as well as mitochondrial apoptosis induction upon JNJ-26481585 treatment. JNJ-26481585-initiated activation of Bax and Bak is not prevented with the addition of zVAD.fmk, suggesting that JNJ-26481585 first disrupts the mitochondria and subsequently activates the caspase cascade. When JNJ-26481585 is used in combination with doxorubicin, we observe not only an increase of proapoptotic Bcl-2 proteins, but also a decrease in the level of the antiapoptotic mitochondrial proteins Bcl-2, Mcl-1, and Bcl-xL. This indicates that Bax, Bak, Bim, and Noxa are crucial for JNJ-26481585-induced as well as JNJ/Dox treatment-induced apoptosis, since RNAi mediated silencing of Bax, Bak, Bim, and Noxa significantly impedes DNA fragmentation upon those treatments.
Furthermore, ectopic overexpression of Bcl-2 profoundly impairs both JNJ-26481585 and combination treatment-mediated apoptosis, abrogates caspase cleavage, and reduces activation of Bax and Bak, underlining the hypothesis that JNJ-26481585 initially targets the mitochondria and then activates caspases.
With the more commonly used HDACI SAHA we confirm the results obtained with the HDACI JNJ-26481585, since combination treatment with SAHA and doxorubicin also induces intrinsic apoptosis, which can be significantly diminished by zVAD.fmk or ectopic overexpression of Bcl-2. Treatment with SAHA and doxorubicin also affects expression levels of pro- and antiapoptotic mitochondrial proteins, thus shifting the balance towards the proapoptotic mitochondrial machinery, resulting in Bax/Bak activation, caspase activation, and subsequently apoptosis.
Taken together, we provide evidence that the HDACIs JNJ-26481585 and SAHA are promising therapeutic agents for the treatment of RMS and that combination regimens with HDACIs represent an efficient strategy to prime RMS cells for chemotherapy-induced apoptosis. These findings have important implications for mitochondrial apoptosis-targeted therapies of RMS.
In der vorliegenden Arbeit wurde ein Flugzeitmassenspektrometer (TOF-MS) für die Messung von halogenierten Spurengasen charakterisiert und das verwendete analytische System optimiert. Ein TOF-MS hat den Vorteil, dass es die volle Masseninformation aufzeichnet. Dadurch ist es möglich, auch im Nachhinein Substanzen zu identifizieren und retrospektiv auszuwerten. Eine retrospektive Auswertung kann helfen, Auswirkungen auf die Atmosphäre besser abschätzen zu können. Aus diesem Grund wurde mit Hilfe des TOF-MS ein digitales Datenarchiv durch regelmäßige Messungen von Luftproben, die am Taunus Observatorium auf dem Kleinen Feldberg genommen wurden, initialisiert. Durch die Wahl des Taunus Observatoriums werden in unmittelbarer Nähe des industriellen Ballungsraums Rhein-Main auf der Nordhemisphäre Luftproben genommen, wodurch die Wahrscheinlichkeit erhöht wird, unbekannte Substanzen in erhöhter Konzentration zu messen.
Bevor das TOF-MS jedoch für die Initialisierung des Datenarchivs verwendet werden konnte, wurde es charakterisiert und mit einem, für die Analyse von halogenierten Kohlenwasserstoffen etablierten QP-MS verglichen. Um beide Detektoren vergleichen zu können, erfolgte die Probenaufgabe, Probenaufkonzentrierung und die Separation der Probe im Gaschromatographen innerhalb eines gemeinsamen Systems. Nach der Separation im GC teilt sich der Trägergasfluss auf. Die Charakterisierung des TOF-MS und der Vergleich mit dem QP-MS umfasst die Auswertung der Daten, die Messpräzision, die Linearität, die Sensitivität der Detektoren, die Massenauflösung und die Massenachsenbestimmungsgenauigkeit.
Hinsichtlich der Messpräzision liegen beide Massenspektrometer, wie ermittelt auf dem selben Niveau, wodurch sie auch sehr geringe Variabilitäten in den Mischungsverhältnissen von halogenierten Kohlenwasserstoffen aufzeichnen können.
Die Linearität der Detektoren ist substanzspezifisch. Während das QP-MS in Übereinstimmung mit bereits literaturbekannter Eigenschaft, einen sehr großen linearen Bereich aufweist, zeigt das hier verwendete TOF-MS für 2/3 aller ausgewerteter Substanzen starke substanz- und fragmentabhängig Nichtlinearitäten. Das nichtlineare Verhalten des Detektors des TOF-MS zeigt sich auch bei den Messvergleichen, wobei jedoch nur signifikante Abweichungen bei sehr hohen und sehr niedrig gemessenen Mischungsverhältnissen beobachtet wurden. Diese starke Nichtlinearität stellt eine große Einschränkung für eine retrospektive Auswertung unbekannter Substanzen dar, da deren Verlauf nur qualitativ nicht aber quantitativ dargestellt werden kann.
Die Massenauflösung liegt beim TOF-MS bei 1000 mit einer Massenachsenbestimmungsgenauigkeit zwischen 50-170~ppm, wodurch es dem QP-MS, welches nur Einheitsauflösung vorweist, weit überlegen ist. Mit dieser Auflösung und Massenachsenbestimmungsgenauigkeit ist das TOF-MS in der Lage einen halogenierten von einem nichthalogenierten Kohlenwasserstoff quantitativ zu trennen.
Zum Vergleich der Sensitivität der beiden Massenspektrometer wurde das QP-MS in drei verschiedenen Modi betrieben: Zum einen dem SCAN-Modus, dem operationalen SIM-Modus, welcher im regulärem Messbetrieb verwendet wird und mehrere Ionen pro Zeitfenster misst, und dem optimierten SIM-Modus, welcher nur ein Ion der jeweiligen Substanz misst. Das TOF-MS hat die gleiche Sensitivität wie das QP-MS im optimierten SIM-Modus. Das TOF-MS hat eine um den Faktor 3 höhere Sensitivität als das QP-MS im operationalen SIM-Modus und eine um den Faktor 12 höhere Sensitivität als das QP-MS im SCAN-Modus bei den betrachteten Substanzen.
Die Initialisierung des digitalen Datenarchivs wurde im Oktober 2013 mit der Probennahme am Taunus Observatorium begonnen, wobei in der vorliegenden Arbeit der Zeitraum von einem Jahr betrachtet wurde. Es wurden Identifizierungen aus regulären Proben der Taunus Observatoriums-Zeitreihe durchgeführt und so die Substanzen HFC-32, HFC-245fa,HCFC-133a und HFO-1234yf gefunden. Zusätzlich stellte Martin Vollmer (Eidgenössische Material und Prüfgesellschaft) zwei Gasmischungen zu Verfügung für die Identifikation von noch nicht am System vermessenen Substanzen. Somit konnte die Vielfalt an diesem System vermessener Substanzen von 40 auf insgesamt 64 Substanzen erweitert werden.
Von den neu identifizierten Substanzen wurden HFC-227ea, HFC-236fa, HFC-32, HCFO-1233zd, HFO-1234zd, HFO-1234yf, HFC-245fa, HCFC-31, HFC-133a, Isofluran und HFC-112 in der Taunus Observatoriums-Zeitreihe gefunden und rückwirkend aufgearbeitet.
Durch die retrospektive Auswertung ist das TOF-MS für seine charakterisierte Anwendung zum Einsatz gekommen.
African trypanosomes cause a parasitic disease known as sleeping sickness. Mitochondrial transcript maturation in these organisms requires a RNA editing reaction that is characterized by the insertion and deletion of U-nucleotides into otherwise non-functional mRNAs. Editing represents an ideal target for a parasite-specific therapeutic intervention since the reaction cycle is absent in the infected host. In addition, editing relies on a macromolecular protein complex, the editosome, that only exists in the parasite. Therefore, all attempts to search for editing interfering compounds have been focused on molecules that bind to proteins of the editing machinery. However, in analogy to other RNA-driven biochemical pathways it should be possible to stall the reaction by targeting its substrate RNAs. Here we demonstrate inhibition of editing by specific aminoglycosides. The molecules bind into the major groove of the gRNA/pre-mRNA editing substrates thereby causing a stabilization of the RNA molecules through charge compensation and an increase in stacking. The data shed light on mechanistic details of the editing process and identify critical parameters for the development of new trypanocidal compounds.
Chemistry and time
(2015)